Indian Journal of Chemistry Vol. 39B, May 2000, pp. 363 - 367

Synthesis and biological activity of 16~-morpholinosteroid derivatives

Poonarn Piplani & Dharam Paul lindal* University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, 160014, India

and

H Fajrak, C Prior & I G Marshall Department of Physiology and Pharmacology, Royal College, University of Strathclyde,

Glasgow GI IXW, Scotland, United Kingdom

Received 15 June 1999, accepted (revised) 1 December 1999

Fusion of morpho line at the 16 position of the steroid J1ucJeus has been carried out to prepare monoquatemary 6,7 and 8 and bisquatemary ammonium compounds 13 and 14. Compounds 13 and 14 partly resemble chandonium iodide in struc­ture. All the compounds have been evaluated for their neuromuscular blocking and ganglion blocking activities. The com­pounds 3, 4 5, II and 12 have been screened for antineoplastic activity at NCI, Bethesda.

Tht: principal use of neuromuscular blocking agents is to provide muscle relaxation to obviate the need for higher doses of volatile anaesthetic agents and making anaesthesia safer and pleasant. Clinically used sux­amethonium chloride, a depolarising neuromuscular blocking agent with transient duration of action, pos­sesses side effects believed to be inherent in its mode of action 1-4

Nondepolarising neuromuscular blocking agents containing benzylisoguinolinium5

-7 and steroid skele­

ton with rapid onset, short duration of action and highly specific for the neuromuscular junction have been developed. The discovery of pancuroniu]ll bro­mide resulted in the development of monoguat~rnary diaminosteroid drugs8

- ' 3. Chandonium iodide (Candocuronium) is another bisquaternary heteroster­oid ' 4,'5. It has interonium distance of 1.029 nm l6

, a powerful nondepolarising blocking activity with rapid onset and short duration of action '5. '7 , ' 8. Structure­activity relationship ' 9 has suggested that interonium distance plays a significant role to compete with ace­tylcholine at the neuromuscular junction . In earlier publications 20-22 many modifications of chandonium iodide have been reported . We report herein the syn­thesis and pharmacology of mono- and bisquaternary aminosteroids, partly resembling chandonium iodide. The interonium distance between two quaternary heads in compound 14 is 1.135 nm as measured by Drieding models.

17-0xo-5-androsten-313-01 1 was brominated using cupric bromide in dry methanol to obtain 16a-bromo-

17-oxo-5-androsten-313-of3-1

5 2. Condensation of the compound 2 with dry morpholine was carried out at boiling temperature to obtain I 613-morpholino-1 7-oxo-5-androsten-313-01 3 (Scheme I). In the NMR spectrum compound 3 showed a multiplet centered at 8 3 .05 (1 H, 16a-H). The assignment of the configura­tion at 16-position in the compound 3 was done as

previously reported24-26

. Sodium borohydride reduc­tion of 3 in methanol at room temperature yie lded 313, 1713-diol 4. Its NMR spectrum showed signals at 8 2.82 (IH, g, .J=9Hz, 16a-H), 3.45 (IH, d, J=9Hz,17a-H) and 3.51 (I H,m, 3a-H). The acetyla­tion of 4 was effected with acetic anhydride in pyri­dine at 100°C to obtain the diacetate 5. Its proton resonance signals appeared at 8 2.03 (3H, s, 313-OCOCH3), 2.10 (3H, s, l713-0COCH3), 3.07 ( IH, g, J=9Hz, l6a-H), 4.58 (I H, m, 3a-H) and 4.80 (I H, d, J=9Hz,17a-H). IR bands at 1732 and 1239 cm- I indi­cated the acetoxy function . Quaternisation of the amines 3 and 4 with methyl iodide in ethanol yielded monoquaternary compounds 6 and 7. 1613-Morpholino-5-androstene-313, 1713-diol diacetate methiodide 8 was prepared from 1613-morpholino-5-androstene-313, 1713-diol diacetate 5 in dichlo­romethane containing methyl iodide at room tem­perature. The monoquaternary compound showed vi­brational C=O stretching at 1730 cm- I in the IR spec­trum.

Oppenauer oxidation of 3 using a cyclohexanone-toluene system afforded 1613-morpholino-4-

364 INDIAN J CHEM, SEC B, MAY 2000

___ >~ --Br

HO HO

(1) (2 )

/ HO

( 3)

./"'N U (10)

o

R

(q R = OH

o II

(5) R=O-C-CH)

(9)

(111 R=OH

o II

(12) R= O-C-CH)

Scheme I

androstene-3 , 17-dione 9 (UV maximum at 238 .6 nm). Compound 9 was refluxed with freshly distilled pyr­rolidine in methanol to obtain the enamine 10 (UV maximum at 274.6 nm). It showed a vinylic proton singlet at () 4.70 (l H,4-H) and 5.0 (I H,6-H), respec­tively. Sodium borohydride reduction of 10 in metha­nol at room temperature gave 16(3-m9rpholino-3(3-pyrrolidino-5-androsten-l '7(3-01 11. A proton reso­nance signal appeared at () 5.30 (lH, m, 6-H). The

amine 11 was acetylated with acetic anhy~ride in pyridine at 100°C to give 16(3-rnorpholino-3 (3-pyrrolidino-5-androsten-17(3-yl acetate U. IHNMR spectra of 12 showed the proton resonance signals at () 2.20 (3H, s, -OCOCH.l), 3.03 (IH, q, J=9Hz,16o.-H)

and 4.78 (l H, d, J=9Hz,17o.-H) . IR spectra showed bands at 1725 and 1230 cm- I for the acetoxy function. Bisquaternary compounds 13 and 14 were prepared by treating the tertiary amines 11 and 12 with methyl

PIPLANI et a/. : SYNTHESIS OF 16P-MORPHOLINOSTEROlD DERrY ATIYES 365

R

(6) R =OH, Rl =(=0)

(7) R=OH R1=OH

o 1/

o

o I

(8) R=O -C-CH)I 1 II

R =O-C-CH ,

(13) R=OH o \I

(14) R= O-C-CH)

iodide in absolute ethanol at refluxing temperature and with dichloromethane at room temperature, re­spectively.

Biological activities

Neuromuscular blocking activity

Compounds 7, 8, 13 and 14 were tested for their neuromuscular blocking activity. Monoquaternary compound 7 was less potent than tubocurarine. Com­pound 13, a bisquaternary compound was found to be more potent than its corresponding monoquaternary mononitrogen analogue.

The bisquaternary 17-acetoxy compound 14 was fou nd to be more potent than the corresponding 17-hydroxy analogue. The compound 14 has been found to be 1.8 times more potent than 13. The neuromus­cular block ing potencies of compounds 13 and 14 were found less than that of tubocurarine. The de­tailed pharmacological profile will be publ ished sepa­rately elsewhere.

Anti neoplastic activity

The compounds 3, 4, 5, 11 and 12 have been screened for' their in vitro antitumor activity against the cell panel consisting of 60 lines at National Can­cer Institute, Bethesda, Maryland, USA. A 48 hr con­tinuous exposure protocol was used and a sulforho­damine B (SRB) prote in assay was used to estimate

cell viability or growth. Selective analysis of the compounds for differential cellular sensitivity based on the response parameters G 150, TGI and LC50 in­dicate that the compounds have statistically insignifi­cant antineoplastic activity for further studies.

Experimental Section Melting points reported are uncorrected. 'HNMR

spectra were recorded on AC-J OOF, 300 MHz, Varian EM-390, 90 MHz and EM-360, 60 MHz NMR in­struments using TMS as an internal standard (chemi­ca l shifts in 8, ppm); IR spectra in KEr (vmax in cm-') on Perkin- Elmer 882 and UV spectra in methanol on a Lamda 15 spectrophotometer models respectively; and mass spectra on a V6-11-250 J 70S. The purity of the compounds was established by TLC and by ele­mental analyses (C,H,N) . Elemental analyses were carried out on a Perkin-Elmer-2400. Anhydrous so­d ium sulphate was used as a drying agent.

16f3-Morpholino-17-oxo-5-androsten-3f3-o1 3. Compound 2 (0.5 g) was refluxed with morpholine for I hr. After removal of maj ority of the morpholine by distillation under reduced pressure, the product was precipitated with water. The product was crystallised from acetone to afford 3 (0.2 g, 49.2% ); mp, 200-204° (Iit24 mp 200°); IR: 1200,1075 ,1725 (C=O); ' H NMR: 0.93 (3H, s, 18-CH3) , 1.06 (3H, s, 19-CH3) , 2.46-2.83 (4H, m, J=5Hz, N- methylenes of morpho lino func­

tion), 3 .05 (rH, m, 16a-CH), 3.63 (5H, t, J= 5Hz; 0 -methylenes of morpholino function , 3a-CH), 5.4 (IH, m, 6-CH) (Found : C, 73.50; H, 9.40 ; N, 3.75 . C23H3sNOJ requires C, 73.95 ; H, 9.44; N , 3.75%).

16f3-Morpholino-S-androstene-3 (3,17(3 -diol 4. Sodium borohydride (1.0 g) was added to a stirred suspension of3 (1.0 g) in methanol ( 100 mL) in small quantities at room temperature. Stirring was continued for 2 hr, the reaction mixture was poured into ice-co ld water (500 mL) and the aqueous uspension was ex­tracted with chloroform (4x 50 mL). T he combined chloroform extract was washed with water, dried and solvent removed under reduced pressure to give a solid residue which was crystallised from methanol to afford 4 (0.7 g, 65.7%); mp 245-48°; IR: 3473 (O-H), 1070 (C-O stretching); ' H NMR: 0.70 (3 H, s, \ 8-CH3), 1.01 (3H, s, \9- CH3), 2 .55-2 .79 (4H , m, J=5Hz, N-methylene of morpholino function), 2.82 (IH, q, J=9H z, 16a- CH), 3.45( IH, d, .I=9Hz, 17a­CH), 3.51 (IH, m, 3a-CH), 3.65-3.77 (4H, m, J=5 Hz, O-methylenes of morpholino function) , 5.30 (IH, d, 6-CH) (Found : C, 73.30; H, 9.88; N , 3.54. C23HJ7 OJ requ ires C, 73.56; H, 9.93; N, 3.73%).

366 INDIAN J CHEM, SEC B, MA Y 2000

16f}-Morpholino-5-androstene-313,17f}-diol di-acetate 5. A mixture of 4 (0.5 g) and acetic anhydride (3 mL) in pyridine (2 mL) was heated on a steam-bath for I hr. The contents were then poured into ice-cold wa­ter (100 mL) and basified with ammonia. The precipi­tate. obtained was filtered, washed with water, dried and crystallised from methanol to afford 5 (0.4 g, 65.4%); mp 208-10°; IR: 1732 (OCOCH3) , 1239 [C­C(=O)-O]; 1160 (C-O stretch); 'H NMR: 0.81 (3H, s,

18-CH3) , 1.03 (3H, s, 19- CH3) , 2.03 (3H , s, 313-OCOCH3) , 2 . 10 (3H, s, 1713-0COCH3) , 3.07 (IH , q, J=9Hz, 16a- CH),3.61-3.71 (4H, m, O-methylenes of morpholino function), 4 .58 (IH, m, 3a-CH), 4.80 (IH, d, J= 9Hz, 17a-CH); 5.30 (IH , m, 6-CH); MS: mlz 459 (M+) (Found : C, 70 .50; H, 9.04; N, 3.02. C27 H4,NOs requires C, 70.55 ; H, 8.99; N , 3.05%).

16f}-Morpholino-17 -oxo-5-and rostcn-3 f}-ol me­

thiodide 6. Methyl iodide (3 mL) was added to a re­fluxin g solution of 3 (0.5 g) in absolute ethanol (20 mL) and the solution was refluxed for 30 min . The

so lvent was removed under reduced pressure and the res idue obtained was crystallised from ethanol to af­ford 6 (0.4 g, 58 .0%); mp 255-60° (Found: C, 55.72; H, 7.39; N , 2.56. C24H38N03 I requires C, 55.92; H, 7.43; N, 2.72%).

16f}-Morpholino-5-androstene-313, 17f}-diol me­

thiodide 7. Methyl iodide (0.5 mL) was added to a refluxing solution of 4 (0.2 g) in absolute ethanol (15 mL) and the solution refluxed for 30 min , processed as usual and the residue obtained was crysta ll ised from acetone to afford 7 (0 .2 g, 58.1%); mp 235-40° (Found: C, 55.40; H, 7.80; N , 3.08. C24H40N03 I re­quires C, 55 .70; H, 7.79; N , 2.71%).

16f}-Morpholino-5-androstene-313,17f}-diol di-

acetate methiodide 8. Methyl iodide (0.4 mL) was added to a solution of 5 (0.2 g) in dichloromethane (5 mL) and allowed to stand at room temperature for two days. The solvent was removed under reduced pres­sure to obtain a solid residue which was crystallised from acetone to afford 8 (0.2 g, 57.25%), mp 238-40° (Found: C, 55 .66; H, 7.23 ; N, 2.53. C28~4NOs I re­quires C, 55.90; H, 7.37; N, 2.33%).

16f}-Morpholino-4-androstene-3,17-dione 9.

1613-Morpholino - 17-oxo-5-androsten -313-013 (0.1 g) was dissolved in a mixture of cyclohexanone (10 mL) and dry toluene (150 mL) and the solution was sub­jected to azeotropic distillation to remove traces of moisture. The distillation was continued at a slow rate during dropwise addition of a solution of aluminium isopropoxide (1.0 g) in dry toluene (20 mL). The re-

action mixture was refluxed for 4 hr and allowed to stand at room temprature for 12 hr. The slurry was filtered and the filtrate was steam distilled until the complete removal of organic solvents was effected. The residual aqueous suspension was allowed to stand at room temperature and extracted with chloroform (4 x25 mL) . The combined chloroform extract was washed with water, dried and the solvent removed to obtain 9 (0.5 g, 50.3%); UV 238.6 (4 .36); IR: 1726 (C=O), 1674 (C=C-C=O) (Found: C, 74 .0 I; H, 8.85 ; N, 3.51. C23H33N03 requires C, 74.36: H, 8.95 ; 3.77%).

1613-Morpholino-3-pyrrolidino-3,5-and rostad ien-17-one 10. Freshly distilled pyrrolidine (2 mL) was added to a refluxing solution of 9 (2.5 g) in methanol ( 15 mL). Refluxing was further continued for 10 min and the solution was concentrated to induce crystal 1 i­sation. The crystalline material was filtered , washed with methanol and dried to afford 10 (2 .5 g, 87.4%); mp 178-80°; UV: 274 (4 . 13); IR: 1741 (C=O): 'H NMR: 0.90 (3H, s, 18-CH3), 1.0 (3H, s, 19-CH3), 2 .93-3.16 (5 H, m, N-methylenes of morpholin o func­tion, 16a -CH), 3 .66 (4H , t, J =5Hz, O-methylenes o f morpholino function), 4.70 (1 H, s, 4-CH), 5.0 (I H, m, 6-CH) (Found: C, 75.90; H, 9.32; , 6. 57. C27 H40N20 2 requires C, 76 .37; H, 9.50; N, 6.60%).

16f}-Morpholino-3f}-pyrrolidino-S-androsten-17f}-

01 11. Sodium borohydride (1.0 g) was added to a stirred suspension of 10 (1.0 g) in methanol (40 mL) in small quantities at room temperature. Stirring was continued for 2 hr, the reaction mixture was poured into ice-cold water (900 mL) and the aqueous suspen­sion was extracted with chloroform (4 x 100 mL). The combined chloroform extract was washed with water, dried and solvent removed under reduced pressure to obtain a solid residue which was crystallised from acetone to afford 11 (0.5 g, 49.6%), mp 188-90°; 'H NMR: 0.73 (3H, s, 18-CH3), 1.03 (3 H, , 19-CH3), 2.33-2.74 (8H, m, N-methylenes of morpholino and pyrrolidino functions), 2.80 (IH, m, 16a-CH),3.40 (IH, q. J=9Hz, 17a-CH),3.50-3 .83 (4H, t, J=5Hz, 0-methylenes of morpholino function), 5.30 (I H, m, 6-CH);[af2-20.43° (c 0.07, CHCh); MS : mlz 428 (M+) (Found: C, 75.21; H, 10.47; N, 6.50. C27 H44N20 2 re­quires C, 75.65; H, 10.35; N, 6.54%).

16f}-Morpholino-3~pyrrolidino-S-andl'osten- 17f}­

yl acetate 12. A mixture of 11 (0.3 g) in pyridine (0 .5 mL) and acetic anhydride (1 mL) was heated on a steam-bath for I hr. The reaction mixture was poured into ice-cold water and basified with ammonia. The precipitated material was filtered and the residue

PIPLANI et al.: SYNTHES IS OF 16P-MORPHOLINOSTEROID DERIVAT IVES 367

crystalli sed from acetone to afford 12 (0.20 g, 60.79%); mp 198-200°; IR: 1725 (OCOCH,), 1230, [C-C(=O)--Ostretch); 11-1 NMR : 0.86 (3 1-1, s, 18-CH,), 1.06 (3 1-1 , s, 19-CH3), 2.20 (3 1-1 , s, -OCOCf-h), 2.40-2.73 (8 1-1 , m, N-methylenes of morpholino and pyrro­lidino functions) , 3.03 (11-1 , q, J=9I-1z, 16a -CII), 3.63 (4 1-1 , m, J =5I-1z, O-methylenes of morpholino func­tion), 4.78 ( 11-1 , d, J =9I-1z, 17a-CH), 5.30 (11-1, m, 6-CH) (Found : C, 73 .61; 1-1 , 9.90; , 5.89. C29 1-146N20 , requires C, 73 .99; 1-1,9.85; N, 5.95%).

16()-Morpholino-3()-pyrrolidino-S-androsten-17()-01 dimethiodide 13. Methyl iodide (0.4 mL) was added to a refluxing so lution of 11 (0.2 g) in abso lute ethanol ( 10 mL) and refluxing was continued for 30 min . The reaction mi xture was cooled, filtered and concentrated to induce crysta ll isation. The crystall ine material was filtered , washed and dried to afford 13 (0 .2 g, 45.2%); mp 282-85° (Found: 'C, 48.60; 1-1 , 7.14;

, 3.51. C29 1-1 soN20 212 requires C, 48.88 ; 1-1 , 7.07; N, 3.93%).

16()-Morpholino-3 ()-pyrrolidino-S-and rosten-17()­yl acetate dimethiodide 14. Methyl iodide (I mL) was added to a so lution of 12 (1 .0 g) in dichlo­romethane (50 mL) and all owed to stand at room temperature for four days. The solvent was removed and residue \vashed with ether, filtered and crysta l­li sed from acetone-ethanol to yield 14 (0.1 g, 75.0%). mp 278-80° (Found: C, 48.23 ; 1-1 , 6.87; , 3.49. C311-1 52N20 312 requires C, 49.34; 1-1 ,6.95; ,3.7 1%).

Acknowledgements

The authors gratefu lly acknowledge the financial support of the CSIR, ew Delhi , and Panacea Biotech Limited, New Delhi. We thank Cipla, Bombay fo r the generous supply of DI-IA acetate and Dr V L Naraya­nan , NCI, Bethesda, USA, for carryi ng out the anti-

neoplastic activity. We also thank Mr S K Sethi for hi s technical assistance.

References I Digby-Leigh M, McCoy D D. Belton M K & Lewis G 13. An­

esthesiology. 18, 1957, 698. 2 Gronert G A, Anesthesiology. 53, 1980,395. 3 Craythorne N W 13, Rottenstein II S & Dripps R D. Anesthe­

siology. 2 1, 1960,59. 4 RidingJE, BrJAnaesth. 47 ,1975, 91. 5 Everett AJ. Lowe LA & Wilkinson S. Chern Commun. 1970,

1020. (: Stl.nlake J 13, Waugh R D, Urwin J, Dewar G H & Coker G

G, Brit J Anaesth. 55 , 1983 ,3S. 7 Hughes R & Chapple D J, Brit J Anaesth. 53, 1981, 31 . 8 Il ewett C L & Savage D S, J Chem Soc C, 1968, 11 34 . 9 Speight T M & Avery G S, Drugs, 4, 1972 163 .

10 Buckett W R, Hewett C L & Savage D S, J Med Chelll , 16. 1973. 111 6.

I I Bowman W C, Trends Pharmacol Sci, I, 1980, 263. 12 Rei lly C S & immo W S, Drugs. 34 , 1987, 98 . 13 Feldman S, Drugs ojToday, 32. 1996. 159. 14 Singh H & Paul D, J Chern Soc Perkin, I, 1974. 1475 . 15 Gandiha A. Marshall I G. PaulO & Singh H, J Pharm Plwr­

macol, 26, 1974, 871 . 16 Palmer R A. Kalam M A, Singh II & Paul D. J Clyst Mol Str.

10, 1980,3 1. 17 Gandiha A, Marshall I G, Paul D. Rodger IW. Scott W &

Singh II , C lin up Pharmacol Physiol. 2, 1975, 159. 18 Il arvey A L. Paul D, Rodger I W & Singh H, J Ph arm Phar­

macal. 28, 1976,6 17. 19 Martin Smith M , in Drug Des ig n, Vo l 2 , ed it ed by E

J Ariens (Academ ic Pre ss, ew York ), 19 7 1, 453 . 20 Abraham J. Jindal D P & Singh H, Indian J Chem, 3113, 1992,

566. 2 1 Abraham J, Jindal D P, Singh H, Patnaik G K & Sri mal R C,

EliI' J Med Chem, 28, 1993, 23 1. 22 Verma A K. Lee C Y, Habtemariam S, Harvey A L & Jindal

o p. Eur J Med Chem, 29, 1994, 33 1. 23 Glazier E R, J Org Chern, 27. 1962, 4397 . 24 Hewett C L & Savage D S, J Chem Soc C, 1966.484. 25 Numazava M & Nagaoka M, J Org Chern, 47, 1982, 4024 . 26 Swenson D C, Duax W L, Numazawa M & Osaway, Acta

Crystallagr Sect B, 1336, 1980, 198 1.